Influenza A virus (IAV) represents a significant burden on global health. This work proposes to generate a library of IAV strains each encoding a siRNA targeting known antiviral host genes with the goal of defining the potency of host factors in the context of an in vivo infection. For these studies, we will use a mouse-adapted influenza A virus that lacks a fully functional NS1 protein, thereby rendering the virus significantly attenuated (mIAV). The specific aims of this proposal are to (1) engineer a mIAV-based siRNA library, (2) perform an in vivo RNAi screen to identify host determinants of IAV replication (3) to characterize the identified host restriction factors. To complete this work, a library of siRNAs was introduced into our mIAV, and the viruses were rescued individually. The viruses were used to infect mice to determine which viruses dominated in the population. Several known antiviral factors were identified in a preliminary screen that includes Trim21, Tlr7, RnaseL, Tbk1 and Irf1. In addition, two factors (Zfp182 and Mapk8) with unknown antiviral activity were identified which are currently being characterized.